CN112094809A - Method for extracting exosome from serum or plasma - Google Patents

Abstract

The invention discloses a method for extracting exosomes from serum or plasma, which is simple and convenient to operate, low in cost, high in extraction efficiency and uniform in exosome size, and comprises the following steps: removing cell debris, ultracentrifugation, filtering supernatant, adding a coagulating agent into the supernatant, ultracentrifugation and PBS (phosphate buffer solution) resuspension and precipitation. Experiments show that the exosomes extracted by the method are uniform in size and mostly distributed in the range of 30-80 nm; the content of the marker protein is high, and part of the hybrid protein can be removed, so that the extraction efficiency of exosomes is improved; and the method for extracting the exosome is simple in operation and less in time consumption.

Description

A method for extracting exosomes from serum or plasma

technical field

The present invention relates to the technical field of biomedicine, in particular to a method for extracting exosomes from serum or plasma.

Background technique

Exosomes, discovered in 1986, are double-layer lipid-membrane vesicles with a diameter of about 30-150nm, which can be produced by various cells in the body such as immune cells, stem cells, cardiovascular cells, and reticulocytes. , platelets, nerve cells and tumor cells are actively secreted, widely distributed in peripheral blood, urine, saliva, milk, ascites, amniotic fluid and other body fluids. Exosomes carry a large number of specific proteins (such as cytokines, growth factors) and functional mRNA, miRNA and other biologically active substances.

Although the function and clinical application of exosomes have been confirmed by many studies, the current simple and efficient methods for purifying exosomes still limit the speed of research progress of exosomes. Currently, the commonly used extraction methods include ultracentrifugation, sucrose density gradient Centrifugation, PEG precipitation, magnetic beads, and membrane affinity column methods, etc. Ultracentrifugation is the gold standard for exosome extraction, but ultracentrifugation is time-consuming and labor-intensive, and the extraction rate is low. Many impurity proteins are precipitated at the same time, and the cost of commercial reagents is high.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention aims to provide a method for extracting exosomes from serum or plasma with simple operation, low cost, high extraction efficiency and uniform size of exosomes.

To achieve the above object, the present invention discloses the following technical contents:

A method for extracting exosomes from serum or plasma, characterized in that the steps are as follows:

1) The serum was centrifuged at 2000×g, 4°C, for 20 min, to remove the cell debris, and the supernatant was retained;

2) Centrifuge the supernatant obtained in step 1) at 30,000 × g, 4°C for 20 min, and carefully aspirate the supernatant for retention;

3) passing the supernatant obtained in step 2) through a filter membrane with a molecular weight cut-off of 3KD to collect the filtrate;

4) Add a precipitant to the filtrate obtained in step 3), mix well, and place it in a 2-8°C environment for incubation for 30-60min; the weight fraction ratio of the precipitant:filtrate is: 3-6:1; Preferable precipitant: filtrate 5:1.

The precipitating agent adopts one or more of PEG2000, heavy water, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), copovidone S-630, carboxymethyl cellulose (CMC) and sodium alginate. mixture.

The mixture of two of the precipitants is: PEG20000 and polyvinyl alcohol (PVA); PEG20000 and polyvinylpyrrolidone (PVP); PEG20000 and copovidone S-630; polyvinyl alcohol (PVA) and carboxymethylcellulose ( CMC); polyvinyl pyrrolidone (PVP) and copovidone S-630; when the mixture of precipitants is made by mixing two precipitants, the mixing ratio of the two precipitants is 50:1~1:50;

The mixture of three precipitants is: PEG20000, polyvinyl alcohol (PVA) and copovidone S-630;

PEG20000, polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP);

Polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP) and copovidone S-630;

Polyvinyl alcohol (PVA), copovidone S-630 and carboxymethyl cellulose (CMC);

The mixing ratios of the three precipitants are listed as 1:1:1~1:15:15; the preferred mixing ratios are 1:1:1, 1:5:5, 1:7:7, and 1:15:15.

5) Place the incubated samples in step 4) at 120,000 × g, 4°C, and ultracentrifuge for 90-120 min, discard the supernatant, and retain the precipitate;

6) The pellet obtained in step 5) was resuspended in 100ul PBS, aliquoted, and stored at -80°C for later use.

The invention further discloses the application of the method for extracting exosomes from serum or plasma in improving the extraction efficiency of exosomes. The experimental results show that when the same volume of serum supernatant is enriched with the same volume of exosome enrichment solution, the concentration of exosomes determined by the traditional ultracentrifugation method is 1.8565µg/µl; The concentration of exosomes was 5.3568 µg/µl.

Beneficial effects obtained by the present invention: through transmission electron microscope observation and analysis, the size distribution of the exosomes extracted by the method of the present invention is uniform, and the background is free of pollution, that is, there is no protein contamination problem, the concentration of the extracted exosomes is high, and the extraction rate is high; The results of Western Blot showed that the exosomes extracted by the two methods contained various protein components, and the expression intensity of proteins with different molecular weights was significantly different. The protein content of the method of the present invention was significantly higher than that of the traditional ultracentrifugation method.

The present invention mainly solves the problems of uneven particle size distribution, protein contamination and low extraction rate of exosomes extracted by traditional ultracentrifugation, and focuses on how to extract high-concentration exosomes. extraction concentration.

Description of drawings

Figure 1 is a transmission electron microscope result of exosomes extracted by traditional ultracentrifugation;

Fig. 2 is the transmission electron microscope result figure of the exosome that the method of the present invention extracts;

3 is an analysis diagram of the detection results of the exosomes extracted by the method of the present invention using a nano-particle size detector;

Figure 4 is an analysis diagram of the detection results of exosomes extracted by traditional ultracentrifugation using a nanoparticle size detector;

Figure 5 is an analysis diagram of Western Blot results.

Detailed ways

The present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments are to be understood as illustrative, rather than limiting, of the scope of the invention, the spirit and scope of the invention being limited only by the claims. For those skilled in the art, on the premise of not departing from the spirit and scope of the present invention, various changes or modifications to the material components and dosages in these embodiments also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are commercially available.

Example 1

Traditional ultracentrifugation:

The serum was transferred to an ultrafiltration centrifuge tube, and the medium was centrifuged at 2000 r/min for 20 min at 4° C. to further remove cells and cell debris to obtain 700 ml of supernatant culture fluid. Particles >200 nm were then removed through a 0.2 μm filter. The exosomes were collected by centrifugation at 34288 r/min for 80 min at 4 °C, and the resulting precipitate was exosomes, and 500 ul of exosome enrichment solution was obtained.

Example

The steps of extracting exosomes from serum or plasma of the present invention:

1) The serum was centrifuged at 2000×g, 4°C for 20min, to remove the cell debris, and obtain 700ml of supernatant culture medium;

2) Centrifuge the supernatant obtained in step 1 at 30,000 × g, 4°C for 20 min, and carefully aspirate the supernatant for retention;

3), pass the supernatant obtained in step 2 through a filter membrane with a molecular weight cut-off of 3KD, and collect the filtrate;

4) Add a precipitant (precipitant: filtrate 5:1) to the filtrate obtained in step 3, mix well, and incubate at 2-8°C for 30-60min;

5) Place the incubated samples in step 4 at 120,000×g, 4°C, ultracentrifuge for 90-120min, discard the supernatant, and keep the precipitate;

6) Resuspend the pellet obtained in step 5 with 100 ul PBS to obtain exosomes, and obtain 500 ul of exosome enrichment solution, which is packaged and stored at -80 °C for later use.

The precipitating agent in step 4 adopts one or more of PEG20000, heavy water, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), copovidone S-630, carboxymethyl cellulose (CMC) and sodium alginate mixture.

The precipitating agent can be any one of the precipitating agents described in step 4. The mixture of precipitants can also be any mixture of two precipitants, such as:

PEG20000 and heavy water;

PEG20000 and polyvinyl alcohol (PVA);;

PEG20000 and polyvinylpyrrolidone (PVP);

PEG20000 and copovidone S-630;;

Polyvinyl alcohol (PVA) and copovidone S-630;

polyvinyl alcohol (PVA) and carboxymethyl cellulose (CMC);

Polyvinylpyrrolidone (PVP) and sodium alginate;

Polyvinylpyrrolidone (PVP) and copovidone S-630;

Preferred mixtures of two precipitants are: PEG20000 and polyvinyl alcohol (PVA); PEG20000 and polyvinylpyrrolidone (PVP); PEG20000 and copovidone S-630; polyvinyl alcohol (PVA) and carboxymethylcellulose (CMC);

Polyvinylpyrrolidone (PVP) and copovidone S-630.

When the mixture of precipitants is made of two precipitants, the mixing ratio of the two precipitants is 50:1~1:50; it can be 50:1, 30:1, 25:1, 10:1, 1 : 1, 1:5, 1:10, 1:20; preferably 5:1~1:8; most preferably 2:1~1:5.

Mixtures of precipitants can also be made from three precipitants, such as:

PEG20000, heavy water and polyvinyl alcohol (PVA);

PEG20000, polyvinyl alcohol (PVA) and copovidone S-630;

PEG20000, polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP);

Polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP) and copovidone S-630;

Polyvinyl alcohol (PVA), copovidone S-630 and carboxymethyl cellulose (CMC);

Polyvinylpyrrolidone (PVP), carboxymethylcellulose (CMC) and sodium alginate;

Polyvinylpyrrolidone (PVP), Copovidone S-630 and Carboxymethylcellulose (CMC).

The preferred mixture of three precipitants is: PEG20000, polyvinyl alcohol (PVA) and copovidone S-630;

PEG20000, polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP);

Polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP) and copovidone S-630;

Polyvinyl alcohol (PVA), copovidone S-630 and carboxymethyl cellulose (CMC);

When the mixture of precipitants can be formed by mixing three precipitants, the mixing ratios of the three precipitants are listed as 1:1:1~1:15:15; the preferred mixing ratios are 1:1:1, 1: 5:5, 1:7:7, 1:15:15.

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Discussion of the results:

1. Observation of exosome morphology:

We used transmission electron microscopy (transmission electron microscope hitachiHT7800/HT7700) to directly observe the morphology of exosomes:

1) Use a pipette (Thermo Company, USA) to draw 20ul of the sample and drop it on the carbon film copper mesh for 3-5min, and then use filter paper to absorb the excess liquid.

2) Put 2% phosphotungstic acid on a 150 mesh carbon support film copper mesh for 1-2 minutes, absorb excess liquid with filter paper, and dry at room temperature.

3) Observation under a transmission electron microscope, collecting images for analysis.

2. Particle size detection

We use a Zetasizer Nano ZSP to directly detect the particle size distribution of exosomes:

1) Use a pipette (Thermo Company, USA) to draw 20ul of the sample and drop it in the sample cell for 3-5min.

2) Observe under the nanometer particle size detector and collect images for analysis.

3. Western Blot

1. SDS-PAGE electrophoresis

1.1 Prepare separating gel and stacking gel with appropriate concentration. The specific formula is operated according to the instructions of the SDS-PAGE gel preparation kit (Beijing Soleibo Technology Co., Ltd., P1200);

1.2 Freshly prepare electrophoresis buffer, use 80V electrophoresis (electrophoresis apparatus, Biole, PowerPac HC) until the band runs out of the stacking gel, then increase to 120V until the Loading just runs out of the gel to stop electrophoresis.

2. Film transfer (film transfer instrument, Bole, Trans-Blot SD)

2.1 Take out the gel after electrophoresis, cut off the stacking gel, and put the separating gel into the prepared 1× membrane transfer solution (Solibo, D1060) to equilibrate for 20min;

2.2 Cut the PVDF membrane (Millipore IPVH00010) and thin and thick filter paper of appropriate size, activate the PVDF membrane with anhydrous methanol (Sinopharm Group, 80080418), and then put the PVDF membrane and thin and thick filter paper into the transfer solution for equilibration for 20 minutes;

2.3 Place in the order of negative electrode-thick filter paper-thin filter paper-glue-PVDF membrane-thin filter paper-thick filter paper-positive electrode. Be careful not to generate air bubbles during the placement process. 200mA constant current transfer membrane for 1h.

3. Blocking and Antibody Incubation

3.1 Use 1×TBST (Solebo, T1081) to prepare a 5% nonfat milk powder (Solebo, LP0031B) solution for blocking;

3.2 According to the antibody instructions, add an appropriate amount of diluted primary antibody (Abcam), incubate at 4°C on a shaker overnight, and then wash with 1×TBST for 3 times, 10 minutes each time;

3.3 Put the washed membrane into the diluted secondary antibody, incubate it on a shaker at room temperature for 1 h, and then wash it three times with 1×TBST for 10 min each time.

4. ECL color rendering

4.1 Prepare an appropriate amount of ECL working solution according to the instructions of the ECL color development kit (Solebo, PE0010);

4.2 Drain the membrane washing solution on the PVDF membrane, drop the prepared ECL working solution evenly on the membrane, incubate at room temperature for 5 minutes, then absorb the excess color developing solution, put it into the automatic chemiluminescence image analysis system for imaging, and use The gray value software was used to analyze the results.

4. Using BCA method to detect the concentration of exosomes extracted by traditional ultracentrifugation method and the method of the present invention

The test was carried out according to the instructions of the BCA protein concentration assay kit (Solebo, PC0020).

Observation and analysis of exosome appearance and morphology

As shown in Figures 1 and 2, the exosomes extracted by two different methods were observed by transmission electron microscopy. The exosomes were round or oval vesicles with a diameter of 40-100 nm. The exosomes extracted by the traditional ultracentrifugation method have uneven particle size distribution and low extraction rate; the exosomes extracted by the method of the present invention have uniform size distribution and no background pollution, that is, there is no protein contamination problem, and the extraction rate is high.

Discussion on detection and analysis of exosome nanoparticle size detector

As shown in Figures 3 and 4, the exosomes extracted by two different methods were detected by a nanoparticle size detector, and the diameters of exosomes were distributed in the range of 50-300 nm. The size distribution of the exosomes extracted by the traditional ultracentrifugation method is uneven, and the extraction rate is low; the size distribution of the exosomes extracted by the method of the present invention is uniform, and the extraction rate is high.

Western Blot Image Analysis

The Western Blot results are shown in Figure 5. The 1-3 bands from the left are the WB results of exosomes extracted by the traditional ultracentrifugation method, and the 4-6 bands from the left are the exosomes extracted by the method of the present invention. of WB results. It can be seen from Figure 5 that the exosomes extracted by the two methods contain a variety of protein components, and the expression intensity of proteins with different molecular weights is significantly different. The protein content of the method of the present invention is significantly higher than that of the traditional ultracentrifugation method.

Using the exosomes obtained by the methods described in Example 1 and Example 2, according to the results of the BCA concentration determination kit, the concentration of exosomes determined by the traditional ultracentrifugation method was 1.8565 µg/µl; while the method of the present invention The resulting exosome concentration was 5.3568 µg/µl.

Claims (4)

1. A method for extracting exosomes from serum or plasma, characterized by the following steps:

centrifuging the serum at 2000 Xg and 4 deg.C for 20min, removing cell debris precipitate, and retaining supernatant;

centrifuging the supernatant obtained in the step 1) at 30000 Xg and 4 ℃ for 20min, carefully sucking out the supernatant and reserving the supernatant;

passing the supernatant obtained in the step 2) through a filter membrane with the molecular weight cutoff of 3KD, and collecting filtrate;

adding a precipitating agent into the filtrate obtained in the step 3), uniformly mixing, and incubating for 30-60min at the temperature of 2-8 ℃; the precipitating agent is: the weight percentage ratio of the filtrate is as follows: 3-6: 1; the precipitant is one or a mixture of more of PEG2000, heavy water, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), copovidone S-630, carboxymethyl cellulose (CMC) and sodium alginate;

placing the sample incubated in the step 4) at 120000 Xg and 4 ℃ for ultracentrifugation for 90-120min, removing the supernatant, and keeping the precipitate;

resuspend the pellet obtained in step 5) with 100ul PBS, subpackage, store at-80 ℃ for use.

2. The method for extracting exosomes from serum or plasma according to claim 1, wherein the precipitant in step 4): and 5:1 of filtrate.

3. The method for extracting exosomes from serum or plasma as claimed in claim 1, wherein the mixture of several precipitants in step 4) means a mixture of two precipitants: PEG20000 and polyvinyl alcohol (PVA); PEG20000 and polyvinylpyrrolidone (PVP); PEG20000 and copovidone S-630; polyvinyl alcohol (PVA) and carboxymethyl cellulose (CMC); polyvinylpyrrolidone (PVP) and copovidone S-630; when the mixture of the two precipitants is formed by mixing the two precipitants, the mixing ratio of the two precipitants is 50: 1-1: 50;

the mixture of three precipitants was: PEG20000, polyvinyl alcohol (PVA) and copovidone S-630;

PEG20000, polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP);

polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and copovidone S-630;

polyvinyl alcohol (PVA), copovidone S-630 and carboxymethyl cellulose (CMC);

the mixing ratio of the three precipitants is 1:1: 1-1: 15: 15.

4. Use of the method for exosome extraction from serum or plasma according to claim 1 for increasing exosome extraction efficiency.

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